Proteins have the capacity to induce potentially life-threatening immune responses. This limitation has hindered their widespread use in consumer end-use applications and products. Indeed, this potential to induce immune responses has come to the attention of the U.S. Food and Drug Administration (FDA), resulting in the requirement for immunogenicity testing both prior to and after approval of new protein therapeutics. However, although there are a number of animal models available for assessing immunogenicity, there are no validated methods to discern relative immunogenicity in humans.
Despite these concerns, the immunogenicity of proteins has long been a concern in the enzyme manufacturing industry. Occupational exposure to proteins has been documented to result in sensitization of industrial and laboratory workers. Sensitization to particular proteins is usually assessed by tests such as the skin-prick test that reveals whether an individual has mounted an immune response to the protein.
Indeed, occupational exposure to proteins has been documented to result in sensitization of industrial and laboratory workers. In most settings, sensitization is controlled by reducing the level of airborne protein (See, Sarlo and Kirchner, Curr. Opin. Allergy Clin. Immunol., 2:97-101 [2002]; and Schweigert et al., Clin. Exp. Allergy 30:1511-1518 [2000]). Occupational exposure guidelines have been implemented that control airborne exposure to proteins. These guidelines, which provide the allowable level of exposure to particular proteins have been useful in reducing the overall number of sensitization events occurring in a given industrial setting. When a new protein is to be manufactured, the establishment of occupational exposure guidelines (OEGs) for the new protein is a matter of serious concern. A commonly accepted method to determine these guidelines is the guinea pig intra-tracheal test (GPIT) (See, Sarlo, Fundam. Appl. Toxicol., 39:44-52 [1997]). In this test, guinea pigs are exposed to the test protein via intra-tracheal instillation for a period of about 10-12 weeks. Serum samples from the animals are taken periodically and tested for their levels of antigen-specific antibody by suitable methods known in the art (e.g., passive cutaneous testing (PCA) for IgG1 and by microimmunodiffusion testing (MID) for precipitating IgG). These results are compared to results obtained from a set of guinea pigs tested with control proteins that have known, effective exposure guidelines (e.g., ALCALASE® enzyme, commercially available from Novo). Determination of serum titers, MID positivity and time to response are considered, and a relative potency value is determined. This method has been used successfully to set OEGs for a number of industrial enzymes.
However, while the GPIT test is useful, it is time consuming and expensive, requiring a number of animals and multiple rounds of testing. Relatively recently, a mouse-based test was established that is reported to reproduce the results obtained in the GPIT, through the use of a less expensive and less cumbersome animal model. The mouse intranasal test (MINT; See, Robinson et al., Toxicol. Sci. 43:39-46 [1998]) is used by some companies to set OEG guidelines. However, industry-wide acceptance has not been achieved for this model (for reviews of predictive tests for protein allergenicity, see Robinson et al., supra, as well as Kimber et al., (Kimber et al., Fundam. Appl. Toxicol., 33:1-10 [1996]; and Kimber et al., Toxicol. Sci., 48:157-162 [1999]).
Thus, although animal models are useful, they have limitations. The use of partially outbred guinea pigs in the GPIT necessitates the use of large numbers of animals in order to achieve statistical significance when comparing responses between groups. In addition, inter-experiment variation in control animal responses is very high, which makes potency determinations based on a single set of control responses less convincing. The MINT assay does not suffer from as much variability in antibody responses because the mice used are typically BDF1 mice, a cross between two highly inbred mouse strains. While this additional level of control allows for more robust data analyses, different strains of mice typically return very different potency rankings for similar enzymes (See, Blaikie, Food Chem. Toxicol., 37:897-904 [1999]; and Blaikie and Basketter, Food Chem. Toxicol., 37:889-896 [1999]). This is likely due to the specificity of the immune response in a mouse line that is been inbred to express very limited MHC molecules. In addition, while data from an individual lab using the MINT assay may be robust, the MINT assay is also plagued by inter-laboratory differences.
Significantly, all animal tests suffer from the inability to provide a suitable representation of the immune response to a given protein in humans. Inbred strains of mice present peptide molecules with the specificity conferred by their murine MHC molecules. Human HLA molecules, while highly related to mouse MHC molecules, do not have identical peptide specificities. Furthermore, inbred mouse strains have been selected for expression of a single I-A and/or I-E molecule, a situation that very rarely occurs in the highly outbred human population. In addition, the mouse immune system has a number of properties which are not found in humans (e.g., the Th1 versus Th2 paradigm that has been described in mice is much less clear in humans). For example, in humans, there is plasticity in Th1 and Th2 phenotypes that can be explained by a genetic inconsistency in the IFN-alpha gene. In contrast, in mice, the Th1 and Th2 phenotypes are not dynamic, due to an insertion in the IFN-alpha gene in these animals (See, Farrar, Nat. Immunol., 1:65-69 [2000]). In addition, humans express HLA class II molecules on activated T cells, while mice do not. Furthermore, human donors typically carry endogenous viruses, and often have subclinical infections, while laboratory mice are typically maintained in a specific-pathogen free (SPF) environment. Another concern is that the C57Bl/6 mouse strain, a popular background for the creation of transgenic mouse models, carries a defined antigen-processing defect that makes comparisons to human derived data of questionable reliability (Kim and Jang, Eur. J. Immunol., 22:775-782 [1992]). Human HLA transgenic mice have become available for application to the mechanistic study of human immune responses (See, Boyton and Altmann, Clin. Exp. Immunol., 127:4-11 [2002]; Black et al., J. Immunol., 169:5595-5600 [2002]; Raju et al., Hum. Immunol., 63:237-247 [2002]; and Das et al., Rev. Immunogenet., 2:105-114 [2000]). However, the use of these animals is limited, as HLA transgenic mice suffer from species-specific immune system complexities. In addition, at least some of the methods used to construct these mice do not allow for accurate analysis of peptide-specific responses, as expression of the HLA transgenes is not correctly regulated. HLA transgenic mice are often used for mapping studies when expressing a single HLA molecule, a situation not found in humans. This is especially of note for HLA-DQ transgenic mice where cross-pairing between different HLA-DQ alleles has been shown to create new peptide presentation specificities (See, Krco et al., J. Immunol., 163:1661-1665 [1999]). Thus, despite advances in the determination, assessment, and comparisons of the immunogenicity of proteins, there remains a need in the art for simple, reliable and reproducible methods to make such determinations.
Likewise, the application of proteins to therapeutic, industrial and nutritional uses is limited by the potential for inducing or exacerbating deleterious immune responses. This potential is especially of concern for the use of recombinant human-derived proteins. Indeed, recombinant human-derived proteins have been demonstrated to induce immune responses directed at self-proteins, resulting in the development of autoimmunity (Li et al., Blood 98:3241-3248 [2001]; and Casadell et al., N. Eng. J. Med., 346:469-475 [2002]). Subsequent reactivation of the immune system after unintended induction of immune responses to industrial or food proteins can be minimized by avoidance. However, this is not the case with human-derived therapeutic proteins. The selection and/or creation of reduced immunogenic protein variants is therefore necessary to improve safety and efficacy of administered proteins. The selection of a naturally occurring hypo-immunogenic protein isomer is an option where several related molecules with similar activities exist. Unfortunately, this is not an option for many therapeutic proteins. Thus, there is a long-felt need in the art for means to produce hypo-immunogenic proteins suitable for use as therapeutics and for other applications.